1887

Abstract

Quantitative real-time PCR (qPCR) on serum provides significant added value to the diagnosis of Q fever, mainly at the acute stage of the disease in seronegative patients and in patients with endocarditis. We evaluated the benefits of DNA concentration in serum by lyophilization to improve qPCR sensitivity. The detection limit of qPCR was determined by comparing six 10-fold dilutions of serum (calibrated with 10 bacteria ml) with and without lyophilization. We also tested, after lyophilization, 73 sera from patients with acute Q fever and 10 sera from patients with endocarditis for which specific qPCR for performed under our usual conditions remained negative. Lyophilization of DNA was found to improve sensitivity of the qPCR; the limit of detection of DNA by qPCR was 100-fold lower in lyophilized sera (1 bacterium ml) than in non-lyophilized sera (10 bacteria ml). Among the 73 sera from patients with acute Q fever, 26 (36 %) were positive after lyophilization, demonstrating a sensitivity gain of 44 % for early negative sera and 30 % for positive sera compared to our usual qPCR conditions. Sensitivity was also higher in sera from patients with endocarditis for which 8/10 (80 %) were positive after lyophilization. Our results serve as a proof of concept that lyophilization increases the sensitivity of qPCR in serum by concentrating bacterial DNA. This technique may be applied for the earlier diagnosis of other fastidious bacteria or viruses and extended to other clinical samples.

Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.000253
2016-06-01
2019-10-23
Loading full text...

Full text loading...

/deliver/fulltext/jmm/65/6/462.html?itemId=/content/journal/jmm/10.1099/jmm.0.000253&mimeType=html&fmt=ahah

References

  1. Al-Soud W. A. , Jönsson L. J. , Radström P. . ( 2000;). Identification and characterization of immunoglobulin G in blood as a major inhibitor of diagnostic PCR. . J Clin Microbiol 38: 345–350.[PubMed]
    [Google Scholar]
  2. Bakken J. S. , Dumler J. S. . ( 2006;). Clinical diagnosis and treatment of human granulocytotropic anaplasmosis. . Ann N Y Acad Sci 1078: 236–247. [CrossRef] [PubMed]
    [Google Scholar]
  3. Calgua B. , Rodriguez-Manzano J. , Hundesa A. , Suñen E. , Calvo M. , Bofill-Mas S. , Girones R. . ( 2013;). New methods for the concentration of viruses from urban sewage using quantitative PCR. . J Virol Methods 187: 215–221. [CrossRef] [PubMed]
    [Google Scholar]
  4. Donatin E. , Drancourt M. . ( 2012;). Optimized microbial DNA extraction from diarrheic stools. . BMC Res Notes 5: 702–707. [CrossRef] [PubMed]
    [Google Scholar]
  5. Dupont H. T. , Thirion X. , Raoult D. . ( 1994;). Q fever serology: cutoff determination for microimmunofluorescence. . Clin Diagn Lab Immunol 1: 189–196.[PubMed]
    [Google Scholar]
  6. Edouard S. , Million M. , Lepidi H. , Rolain J. M. , Fournier P. E. , La Scola B. , Grisoli D. , Raoult D. . ( 2013;). Persistence of DNA in a cured patient and positive culture in cases with low antibody levels bring into question diagnosis of Q fever endocarditis. . J Clin Microbiol 51: 3012–3017. [CrossRef] [PubMed]
    [Google Scholar]
  7. Edouard S. , Million M. , Royer G. , Giorgi R. , Grisoli D. , Raoult D. . ( 2014;). Reduction in incidence of Q fever endocarditis: 27 years of experience of a national reference center. . J Infect 68: 141–148. [CrossRef] [PubMed]
    [Google Scholar]
  8. Eldin C. , Angelakis E. , Renvoisé A. , Raoult D. . ( 2013;). Coxiella burnetii DNA, but not viable bacteria, in dairy products in France. . Am J Trop Med Hyg 88: 765–769. [CrossRef] [PubMed]
    [Google Scholar]
  9. Eldin C. , Raoult D. . ( 2015;). Moving from Q fever to C. burnetii infection. . Epidemiol Infect 26: 1–2.
    [Google Scholar]
  10. Fenollar F. , Fournier P. E. , Raoult D. . ( 2004;). Molecular detection of Coxiella burnetii in the sera of patients with Q fever endocarditis or vascular infection. . J Clin Microbiol 42: 4919–4924. [CrossRef] [PubMed]
    [Google Scholar]
  11. Fournier P. E. , Marrie T. J. , Raoult D. . ( 1998;). Diagnosis of Q fever. . J Clin Microbiol 36: 1823–1834.[PubMed]
    [Google Scholar]
  12. Fournier P. E. , Raoult D. . ( 2003;). Comparison of PCR and serology assays for early diagnosis of acute Q fever. . J Clin Microbiol 41: 5094–5098. [CrossRef] [PubMed]
    [Google Scholar]
  13. Glazunova O. , Roux V. , Freylikman O. , Sekeyova Z. , Fournous G. , Tyczka J. , Tokarevich N. , Kovacava E. , Marrie T. J. , Raoult D. . ( 2005;). Coxiella burnetii genotyping. . Emerg Infect Dis 11: 1211–1217. [CrossRef] [PubMed]
    [Google Scholar]
  14. Haese A. , Huland E. , Graefen M. , Hammerer P. , Noldus J. , Huland H. . ( 1999a;). Ultrasensitive detection of prostate specific antigen in the followup of 422 patients after radical prostatectomy. . J Urol 161: 1206–1211. [CrossRef] [PubMed]
    [Google Scholar]
  15. Haese A. , Huland E. , Graefen M. , Huland H. . ( 1999b;). Supersensitive PSA-analysis after radical prostatectomy: a powerful tool to reduce the time gap between surgery and evidence of biochemical failure. . Anticancer Res 19: 2641–2644.[PubMed]
    [Google Scholar]
  16. Higuchi R. , Dollinger G. , Walsh P. S. , Griffith R. . ( 1992;). Simultaneous amplification and detection of specific DNA sequences. . Bio/Technology 10: 413–417. [CrossRef]
    [Google Scholar]
  17. Higuchi R. , Fockler C. , Dollinger G. , Watson R. . ( 1993;). Kinetic PCR analysis: Real-time monitoring of DNA amplification reactions. . Bio/Technology 11: 1026–1030. [CrossRef]
    [Google Scholar]
  18. Maurin M. , Raoult D. . ( 1999;). Q fever. . Clin Microbiol Rev 12: 18–53.
    [Google Scholar]
  19. Million M. , Walter G. , Thuny F. , Habib G. , Raoult D. . ( 2013;). Evolution from acute Q fever to endocarditis is associated with underlying valvulopathy and age and can be prevented by prolonged antibiotic treatment. . Clin Infect Dis 57: 836–844. [CrossRef] [PubMed]
    [Google Scholar]
  20. Million M. , Bellevegue L. , Labussiere A.-S. , Dekel M. , Ferry T. , Deroche P. , Socolovschi C. , Cammilleri S. , Raoult D. . ( 2014;). Culture-negative prosthetic joint arthritis related to Coxiella burnetii . . Am J Med 127: 786.e7–78786. [CrossRef]
    [Google Scholar]
  21. Morel A.-S. , Dubourg G. , Prudent E. , Edouard S. , Gouriet F. , Casalta J.-P. , Fenollar F. , Fournier P. E. , Drancourt M. , Raoult D. . ( 2014;). Complementarity between targeted real-time specific PCR and conventional broad-range 16S rDNA PCR in the syndrome-driven diagnosis of infectious diseases. . Eur J Clin Microbiol Infect Dis 34: 561–570.[CrossRef]
    [Google Scholar]
  22. Raoult D. , Fournier P. E. , Fenollar F. , Jensenius M. , Prioe T. , de Pina J. J. , Caruso G. , Jones N. , Laferl H. , other authors . ( 2001;). Rickettsia africae, a tick-borne pathogen in travelers to sub-saharan africa. . N Engl J Med 344: 1504–1510. [CrossRef] [PubMed]
    [Google Scholar]
  23. Raoult D. . ( 2012;). Chronic Q fever: expert opinion versus literature analysis and consensus. . J Infect 65: 102–108. [CrossRef] [PubMed]
    [Google Scholar]
  24. Rapp D. , Waller J. , Brightwell G. , Muirhead R. W. . ( 2010;). Lyophilization prior to direct DNA extraction from bovine feces improves the quantification of Escherichia coli O157:H7 and Campylobacter jejuni . . Appl Environ Microbiol 76: 1686–1688. [CrossRef] [PubMed]
    [Google Scholar]
  25. Socolovschi C. , Angelakis E. , Renvoisé A. , Fournier P. E. , Marié J. L. , Davoust B. , Stein A. , Raoult D. . ( 2011;). Strikes, flooding, rats, and leptospirosis in Marseille, France. . Int J Infect Dis 15:, e710e715. [CrossRef] [PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.000253
Loading
/content/journal/jmm/10.1099/jmm.0.000253
Loading

Data & Media loading...

Supplements

Supplementary File 1



PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error